New 16-oxygenated steroids



United States Patent The invention relates to a process for theoxygenation of steroid compounds.

The process according to the invention is characterized in that by achemical or microbiological method l6-oxygenated steroid compounds areprepared of the general formula:

in which R =H or a methyl group R =H(,BOH) or H(}8O Acyl), 0, alkyl(60H), or alkyl R and R together a l6,17-aceta1-, or -ketal group.

The compounds according to the invention are important on account oftheir biological activity.

From pharmacologicai experiments it has appeared that the presentcompounds have i.a. an oestrogenic and uterotropic activity. I

They can be obtained in various manners starting from other steroidcompounds. One of these methods is characterized in that A-3-ketoandrostene compound or a A -3-keto-19-nor-androstene compound isbrought into contact with an in lop-position oxidising fungus or anoxidising enzyme system obtained therefrom.

For the l6-oxygenation of the present steroids fungi of various orderscan be applied. In particular the fungi belonging to the orderMoniliales have appeared to be quite suitable and that especially thegenus Curvularia, such as C. lunata, the order Sphaeriales, such as thespecies Mycosphaerella fragarz'ae and Mycosphaerella latebrosa, theorder Melanconiales, such as the species Colletolrichum phomoiaes andCollezotrichum lindemuthianum and the group Mycelia of the FungiImperfecti, such as the species Sclerotium cofleicolum.

The process according to the invention is carried out in the mannersknown for analogous microbiological conversions by bringing thesubstrate, in conditions suitable for that purpose, into contact withthe enzyme system of the relative fungus. For that purpose first aculture of the relative fungus is, for example, allowed to develop in aculture medium under aerobic conditions, after which a fermentationmedium containing the steroid to be modified, is subjected to thebio-oxygenating action of the mycelium formed.

The culture medium chiefly consists of a carbon and nitrogen source, forexample, a carbohydrate, such as glucose, maltose or starch and anorganic nitrogen source, such as corn steep liquor, yeast extract, meatextract or an inorganic nitrogen source, such as ammonium salts oralkali metal nitrates.

Furthermore an anti-foaming agent, such as glycerylmentation medium,which contains the steroid to be oxygenated and one or more of theabove-mentioned nutrients.

The temperature employed in the microbiological oxidation of the steroidis usually between and 28 C., although higher or lower temperatures, forexample those between 15 and 45 C., are suitable.

The time required for the oxidation of the steroid varies widely, butusually an oxygenation period of from 10 to 72 hours is suihcient.

The 16B-hydroxy compound obtained after completion of the fermentationprocess can be obtained from the fermentation medium in one of thecommon man-.

ners, for example by chromatography, extraction or combinations thereof.

The compounds according to the invention can also be I prepared fromother steroids by a chemical method.

One of the methods is characterized in that a S-ether of l-epi-oestriolor a 16,17-derivative thereof is taken as starting material, whichcompound is reduced to the 16/3-nydroxy-motor-testosterone or a16,17-derivative thereof by means oi an alkali metal in liquid ammoniaby the method described by A. I. Birch in J. Chem. Soc.,

'- page 367 (1950).

Another chemical method is characterized in that a. l7-keto compound ofthe androstane' or l9-noradrostane series is taken as starting materialand converted into the corresponding lo-oximino compound, which byreduction, with for example zinc and acetic acid, is

reduced to a l6-keto compound, which latter compound" is reduced againto the desired l6B-hydroxy compound with, for example, hydrogen in thepresence of a platinum.

catalyst.

Another chemical method is characterized in that a 17-keto-steroid isenolacylated, whereupon the thus obtained A -17-acyloxy compound istreated with lead tetra-acetate to form the corresponding16,6-acyloxy-I7- keto-steroid. By reduction with, for example, lithiumaluminium hydride the 165,175-dihydroxy-stroid compound is obtained fromit.

The l6fl-acyloxy-l7-keto compound obtained can also i first be convertedinto the corresponding 17-hydroxy-17- alkyl compound by an additionreaction and after that into the corresponding lfi-hydroxy compound byhydrolysis.

If desired, the thus obtained steroid compounds can be converted intothe functional derivatives thereof, es-

pecially to the 16,17-diesters, or l7-mono-esters if a 16-mono-stearate, is in most cases also added to theferdecylic acid, lauricacid, palmitic acid, stearic acid, un-

decylenic acid, oleic acid, hexahydrobenzoic acid, cyclopentylpropionicacid, cyclohexylbutyric acid, phenyl acetic acid, phenyl propionic acid,succinic acid, glutaric acid, tartaric acid and carbamic acid.

The esteriiication can be carried out by any method known per se, forexample by reaction with the acid anhydride or the acid halide,preferably in the presence of a tertiary base, such as pyridine orquinoline and, if desired, in the presence of a suitable solvent, suchas ether,

dioxane or benzene.

The 16,17-acetals, or 16,17-ketals of the present ketone or acetophenoneunder anhydric conditions and inthe presence of a catalyst, such ascopper sulphate, hydrogen chloride gas or perchloric acid.

The invention is illustrated by the following examples.

Example I A culture medium containing 20 g. of evaporated corn steepliquor and 20 g. of glucose per 1,000 ml. of distilled Water was, afteradjustment at pH 6.5 (sodium hydroxide solution) and sterilisation for20 minutes at a temperature of 120 C., inoculated with a culture ofMycosphaerella latebrasa, grown on malt agar, per portion of 500 ml. inshaking fiasksot 2,000 ml. content.

These inoculated cultures were shaken X24 hours at 26 C. on a rotatingshaking machine (300 rev/min), after which the mycelium formed waspassed from two shaking flasks into a 30 1. stainless steel fermentationtank, fitted with stirrer and aerator, containing the followingfermentation medium sterilised under the above conditions:

Corn steep liquor (calculated on the basis of dry substance) g 75 Gucose g 75 Anti-foaming oil g 15 Distilled water to L 15 pH=6.5 (sodiumhydroxide solution).

After 48 hours stirring and exposure to air (number of revolutions 200/min; air supply 101./min.) at 26 C., 6 g. of 19-nor-testosteronesuspended in 100 ml. of sterile water were added to the fermentationmedium under aseptic precautions. After that fermentation was continuedfor another 72 hours and then stopped. The thus obtained fermentationliquid was freed from the mycelium by filtration and the clear culturefiltrate extracted with 4 x 4,000 ml. methyl isobutyl ketone.

Then the extract Was evaporated in vacuo to 750 ml. and then washed withrespectively 75 ml. of NaOH-solution 0.1 N, 75 ml, of distilled water,75 ml. of hydrochloric acid 0.1 N and 3 times 75 ml. of distilled Water,after which the thus treated extract was entirely evaporated to drynessin vacuo.

The solid residue (4.05 g.) was then subjected to a countercurrentextraction (toluene/50% methanol 1:1)

to obtain after 96 transports a complete separation of the oxygenatedsteroid. The contents of thetubes 25 to inclusive were evaporated toobtain 2.01 g. of crystalline product with a melting point of l49-151 C.This product was purified by crystallisation from methyl isobutylketone. This treatment yielded 1.44 g, of pure 163-hydroxy-19-nor-testosterone with a melting point of 154-155 C.

R -values:

System toluene-70% methanol (Whatman No. 1):

0.50. System chloroform-formamide (Whatman No. 1):

0.5 g. of l-epi-oestriol is dissolved in 50 ml. of methanol to which 10ml. of 2 N NaOH are added.

Then 0.95 ml. of dimethyl sulphate in 5 ml. of ethanol are addeddropwise in 10 minutes. Next the mixture is refluxed for 5 /2 hours.Then 1 g. of NaOH, dissolved in 100 ml. of water, is added, whereuponthe mixture is cooled to room temperature and the precipitate isfiltered off. The residue is washed with 2 N NaOH and with water andthen recrystallised from methanol. Yield 350 mg. The filtrate is shakenout with chloroform, the chloroform layer Washed with 2 N NaOH, nextwith water until neutral reaction and finally dried and evaporated. Fromit another 35 mg. of substance were recovered.

300 mg. of the thus obtained 3-methyl ether of 16- epi-oestriol aredissolved in 7 ml. of dioxane and 7 ml. of tetra-hydrofurane and slowlyadded dropwise, while stirring, to a solution of 2.5 g. of lithium in200 ml. of liquid ammonia. After the addition of the lithium stirring iscontinued for 30 minutes. 7 The temperature is kept at -60 C. Theammonia is evaporated, to the residue 100 ml. of water are added and themixture is shaken outwith chloroform. The organic layer is separated,washed with Na CO and with water; dried and evaporated to dryness. Theresidue is dissolved in 30 ml. of tetrahydrofurane and after adding 7ml. of 2 N hydrochloric acid refluxed for one hour. After that ml. ofice water are added and extraction takes place with chloroform, theorganic layer is Washed with Na CO -solution and with water, dried andevaporated until dry. The residue is chromatographed over aluminiumoxide and recrystallised from acetone-hexane. Yield 208 mg. of16/8-hydroxy-19-nortestosterone. Melting point 154-156? C,

Analysis.-Found 74.57% C; 9.01% H. Calculated for CmHg Oa I C; [DL]D: ochloroform) Example 111 50 mg. 16fi-hydroxy-19-nor-testosterone aredissolved in 14- ml. of acetone; while stirring vigorously 1 drop of 65%perchloric acid solution is added. The mixture is stirred for 10 minutesat room temperature, after which Example IV mg. of16,6-hydroxy-l9-nor-testosterone are dissolved in 1 ml. of pyridine and0.6 ml. of acetic anhydride. The solution is kept at room temperaturefor one night, after which it is poured into ice; To the aqueous mixture2 ml. of 4 N HCl are added and the mixture is kept at room temperaturefor two hours. tion takes place with chloroform. The organic layer iswashed with a cold Na CO -solution and with water. The solution is driedand the solvent evaporated until dry. Yield 100 mg. of l65,17fidiacetoxy-A -3-keto-19-norandrostene.

Analogously the 16,17-diesters are prepared, derived from trimethylacetic acid, cyclopentyl propionic acid, phenyl propionic acid, capricacid, palrnitic acid and succinic acid.

Example V A culture medium consisting of 5 g. of malt extract, 10 g. ofglucose, 10 g. of soya-meal, 5 g. of sodium chloride, 5 g. of evaporatedcorn steep liquor, 1. g. of calcium carbonate and completed with tapwater to 1 litre, is sterilised for 30 minutes at a temperature of C.and then inoculated with a culture of Curvularia lunata (Ramoena.strain).

This culture is shaken on a shaking apparatus 120 revolutions/minute)for 5 days at 24 C., after which 10 ml. of a solution of A-3-keto17a-methy1-17B-hydroxyandrostone in acetone are added to 1 litreof culturemedium. The solution is incubated for 80 hours at atemperature of 24 C., after which the culture medium is filtered and themycelium washed with acetone. After that filtrate and After that extracIn accordance withv the process described above thel7u-allyl-l9-nor-testosterone is converted into the correspondinglofi-hydroxy-compound by means of Colleto zrz'chum phomoides and the A-3-keto-l7oa-propyl-17B-hydroxy-androstene to the correspondingl6fi-hydroxy-compound by means of Sclerotium cofleicolum.

Example VI To a solution of 0.75 g. of A -3-hydroxy-17-keto-androstenein 10 ml. of isopropenyl acetate 0.5 ml. of sulphuric acid are added,after which the solution is boiled for two hours. Next another 10 ml. ofisopropenyl acetate and 0.5 ml. of sulphuric acid are added, after whichthe reaction mixture is boiled for another two hours and at the sametime evaporated to a small volume. The solution is then diluted withether, the ether layer is separated, washed With a sodium carbonatesolution and water, dried on sodium sulphate and then evaporated untildry. The residue is recrystallised from a mixture of acetone andpetroleum ether to obtain the M' -3,17-diacetoxy-androstadiene.

To a solution of 1 g. of this compound in ml. of glacial acetic acid and1 ml. of acetic anhydride is added 1.25 g. of lead tetra-acetate. Thesolution is stirred for 24 hours at room temperature and then evaporatedin vacuo until dry. The residue is dissolved in benzene, washed with asodium bicarbonate solution and with water, dried on sodium sulphate andevaporated until dry. The residue is recrystallised from petroleum etherto obtain the A 8 8,l6,8-diacetoxy-l7-keto-androstene.

This compound is dissolved in 75 ml. of ether and added to a suspensionof mg. of lithium aluminium hydride in ml. of ether. The suspension isrefluxed for 90 minutes, after which ethyl acetate is added to themixture. The ether solution is washed with water, dried on sodiumsulphate and evaporated until dry. The residue is recrystallised fromaqueous methanol to obtain the A 6 8,l6B,17fl-trihydroxy-androstene.

To a warm solution of 1 g. of this compound in a mixture of ml. oftoluene and 5 ml. of cyclohexanone is added dropwise a solution of 6 ml.of aluminium isopropoxide, in 10 ml. of toluene. The solution isrefluxed 5 for half an hour, after which asolution of 4 g. of sodiumpotassium tartrate in 15 m1. of water is added to it. The mixture issubjected to a steam distillation and then extracted with chloroform.The chloroform layer is washed with a NaCl-solution, dried on sodiumsulphate and evaporated in vacuo until dry. The residue is crystallisedfrom methyl isobutyl ketone to obtain the A -3-keto-l6fl,

17,8-dihydroxy-androstene.

We claim: 1. New steroids of the general formula:

CHa

25 in which P and Q are selected from the group consisting of hydrogen,methyl and ethyl.

2. The new compound of the formula:

Drill et al.: Recent Progress in Hormone Research (1958), vol. 14, pages26-76 (page 61 relied on).

Bernstein et al.: J.A.C.S., vol. 81, page 4573, Sept. 5, 1959.

Allen et al.: I.A.C.S., vol. 81, pages 49684979, Jan.

1. NEW STEROIDS OF THE GENERAL FROMULA: